CN102653729B - Culture medium used for Chinese hamster ovary cells - Google Patents
Culture medium used for Chinese hamster ovary cells Download PDFInfo
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- CN102653729B CN102653729B CN201110051952.2A CN201110051952A CN102653729B CN 102653729 B CN102653729 B CN 102653729B CN 201110051952 A CN201110051952 A CN 201110051952A CN 102653729 B CN102653729 B CN 102653729B
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- Prior art keywords
- hamster ovary
- chinese hamster
- cell
- medium
- sodium
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Abstract
The invention discloses a basal culture medium used for Chinese hamster ovary cells. Based on the total volume of the basal culture medium, the basal culture medium comprises the following basic ingredients: 6-6.5g/L of sodium chloride, 0.5-0.5g/L of potassium chloride, 0.462-1g/L of pyruvic acid sodium, 0.288-0.4g/L of calcium nitrate, 1.464-2g/L of sodium bicarbonate, 5.16-15g/L of D-glucose and 0.156-0.7g/L of disodium hydrogen phosphate. The invention also discloses a fed-batch medium used for Chinese hamster ovary cells and a cultural method of Chinese hamster ovary cells.
Description
Technical field
The present invention relates to field of cell culture.More specifically, the present invention relates to substratum and the corresponding cultural method for Chinese hamster ovary cell (Chinese Hamster Ovary, CHO) cell cultures.
Background technology
CHO(Chinese Hamster Ovary in eukaryotic cell, Chinese hamster ovary cell) cell is the first-selected system of glycosyl protein production of recombinating at present; Because compared with other expression systems, it has many advantages.At present existing increasing pharmaceutical protein has obtained high efficient expression in Chinese hamster ovary celI, and the medicine that wherein many CHO express is put on market, such as EPO, Enbrel etc.
Widely used in Chinese hamster ovary celI is cultivated is at present serum free medium, because the substratum that contains animal serum has multiple unfavorable factor.Because serum free medium is limited for the promotion functions of the growth of cell, expression, so feed supplement technology has become one of emphasis of each mcroorganism pharmacy corporation competition now.
The training mode that Chinese hamster ovary celI is conventional has batch culture, feeding culture, cultured continuously and perfusion culture, and choosing of operator scheme has important impact to the expression of product.Generally in reactor operation, can select different operator schemes according to the feature of the characteristic of object product and requirement and cell strain.The method that early stage bio-reactor large scale culturing Chinese hamster ovary celI adopts is often batch culture.What at present, pharmaceutical protein was produced extensively employing is that stream adds formula cultivation (Fed-batch culture).It is improved on batch culture basis that stream adds formula cultivation, first pack a certain amount of nutrient solution into reactor, inoculating cell is cultivated, constantly consume at the continuous process of growth nutritive substance of cell, the continuous accumulation of meta-bolites, supplement new nutritive ingredient the certain opportunity at logarithmic phase in system, makes cell further growth metabolism, until whole cultivation finishes rear taking-up product.In the production technique that has obtained at present the biological technology products of FDA approval and publish, what occupy main flow advantage is that stream adds or perfusion culture.
International and domestic have a lot of people to develop various CHO substratum and feed supplement formula, as: the CHO substratum (number of patent application: 200410018258.0) of people's inventions such as the domestic Zhang Li of East China University of Science, Zhang Yuanxing.The substratum that has in the world business to sell, if JRH Biosciences company is the company that specializes in cell cultures base product (cell culture media), is that U.S. FDA is specified biological medicine enterprise-specific substratum.The Invitrogen company serum free medium of also producing, but the cultivation base unit weight of carrying out on a large scale antibody drug protein expression needs is very large, somewhat expensive, such as the price of JRH basic medium is at every liter more than 100 yuans, in cost, account for significant proportion, in China, protein drug producer is also faced with the problem of the enormous expenditure of substratum, and protein yield low is also a very large problem simultaneously.
Therefore, this area is in the urgent need to developing new applicable Chinese hamster ovary celI serum free medium that cultivate, high protein output.
Summary of the invention
Object of the present invention is just to provide a kind of substratum of new cultivation Chinese hamster ovary celI, and a kind of cultural method of Chinese hamster ovary celI is also provided.
In a first aspect of the present invention, a kind of basic medium for Chinese hamster ovary cell is provided, in the cumulative volume of described basic medium, described basic medium contains following fundamental component:
6-6.5g/L sodium-chlor;
0.5-0.5g/L Repone K;
0.462-1g/L Sodium.alpha.-ketopropionate;
0.288-0.4g/L nitrocalcite;
1.464-2g/L sodium bicarbonate;
5.16-15g/L D-Glucose; With
0.156-0.7g/L Sodium phosphate dibasic.
In another preference, described basic medium also contains 10-54g/L amino acid, 1.2-7.0g/L trace element, 0.3-1.6g/L VITAMIN and 3-10g/L yeast extract; Described trace element comprises Cu, Fe, Zn, Mg, Mn, Ni, Na, Se, V, Mo, Sn and Si; Described VITAMIN comprises choline chloride 60, thanomin, DL-alpha-lipoic acid, I-inositol, riboflavin, thiamines, cyanocobalamin, D-VB5 salt, Bio, folic acid, niacinamide, para-amino benzoic acid, putrescine and pyridoxol.
In another preference, in basic medium provided by the invention, described amino acid (g/L) is:
In another preference, in basic medium provided by the invention, described trace element (g/L) is:
In another preference, in basic medium provided by the invention, described VITAMIN (g/L) is:
In another preference, in described basic medium, also contain following material (g/L):
In a second aspect of the present invention, a kind of supplemented medium for Chinese hamster ovary cell is provided, in the cumulative volume of described supplemented medium, described supplemented medium contains following fundamental component:
5.67-10.773g/L sodium-chlor;
4.32-8.208g/L nitrocalcite;
100-190g/L D-Glucose; With
3.24-6.156g/L Sodium phosphate dibasic.
In another preference, described supplemented medium also contains 153-300g/L amino acid, 10-19g/L trace element, 5.5-11g/L VITAMIN and 30-100g/L yeast extract; Described trace element comprises Cu, Fe, Zn, Mg, Mn, Ni, Na, Se, V, Mo, Sn and Si; Described VITAMIN comprises choline chloride 60, thanomin, DL-alpha-lipoic acid, I-inositol, riboflavin, thiamines, cyanocobalamin, D-VB5 salt, Bio, folic acid, niacinamide, para-amino benzoic acid, putrescine and pyridoxol.
In another preference, in supplemented medium provided by the invention, described amino acid (g/L) is:
In another preference, in supplemented medium provided by the invention, described trace element (g/L) is:
In another preference, at supplemented medium provided by the invention, described VITAMIN (g/L) is:
In another preference, in described supplemented medium, also contain following material (g/L):
Linolic acid 0.005292-0.0100548
Yeast extract 30-100.
In a third aspect of the present invention, provide the purposes of above-mentioned basic medium provided by the invention, for cultivating Chinese hamster ovary cell.
In another preference, described basic medium is applicable to the Chinese hamster ovary cell of Tetrahydrofolate dehydrogenase (DHFR) system.
In a fourth aspect of the present invention, provide the purposes of above-mentioned supplemented medium provided by the invention, for cultivating Chinese hamster ovary cell.
In another preference, described supplemented medium is applicable to the Chinese hamster ovary cell of Tetrahydrofolate dehydrogenase (DHFR) system.
In a fifth aspect of the present invention, a kind of cultural method of Chinese hamster ovary cell is provided, described method comprises step:
(a) in basic medium provided by the invention as above, cultivate Chinese hamster ovary cell; With
(b) add supplemented medium provided by the invention as above to cultivate Chinese hamster ovary cell at Growth of Cells to logarithmic phase.
In another preference, described Chinese hamster ovary cell is Chinese hamster ovary suspension cell.
In the step (a) of above-mentioned cultural method provided by the invention, with 0.5x10
6in the initial cultivation of density; In step (b), cultivating 3-5 days cell density 4-7x10
6time give feed supplement.
In another preference, basic medium of the present invention and supplemented medium, be all applicable to the amplification procedure step by step from shaking flask, WAVE reactor to fermentor tank etc.
Accordingly, the invention provides a kind of new applicable Chinese hamster ovary celI serum free medium that cultivate, high protein output.
Brief description of the drawings
Fig. 1 has shown the experimental result of match gold base culture medium culturing Chinese hamster ovary celI; Wherein certain antibody titers (Titer) is 0.4g/L; And rhombus is viable cell density (VCD), and square is survival rate, lower same.
Fig. 2 has shown the experimental result of JRH base culture base Chinese hamster ovary celI; Wherein titre (Titer) is 0.2g/L.
Fig. 3 has shown the experimental result of matching golden supplemented medium cultivation Chinese hamster ovary celI; Wherein titre (Titer) is 1.7g/L, mends the feed supplement of 4% volume.
Fig. 4 has shown the experimental result that JRH supplemented medium is cultivated Chinese hamster ovary celI; Wherein titre (Titer) is 0.8g/L, mends the feed supplement of 10% volume.
Fig. 5 has shown the experimental result that Invitrogen supplemented medium is cultivated Chinese hamster ovary celI; Wherein titre (Titer) is 0.6g/L, mends the feed supplement of 4% volume.
Embodiment
Contriver, through extensive and deep research, has found a kind of serum free medium of new Chinese hamster ovary celI, wherein contains a small amount of yeast extract (DMV USA, Inc. (La Crosse, WI)), and do not contain glutamine, this be there is no special requirement.And in culturing process, carry out feeding culture, add supplemented medium provided by the invention at Growth of Cells to logarithmic phase, can make Growth of Cells obtain better.Complete on this basis the present invention.
The object of the invention is to develop a kind of new for cultivating the substratum of Chinese hamster ovary celI, its culture effect, expressing quantity all will reach the requirement of technical grade large scale culturing, are adapted to the development of Chinese biological pharmacy industry.The Chinese hamster ovary celI growth of culture medium culturing provided by the invention is vigorous, cell density can reach 10
7more than cells/ml, cytoactive is fine.
In substratum provided by the invention, contain all essential materials, only add a small amount of yeast extract, protein content is few, is conducive to separation and purification, is applicable to cultivate on a large scale Chinese hamster ovary celI to express pharmaceutical protein.
The problem of growing in serum free medium in order to solve cell, basic medium provided by the invention, except containing fundamental component, has also added amino acid, trace element, VITAMIN and other composition (as lipid etc.).
(1) amino acid: added each amino acid of the best proportioning of determining by strict experiment, really made the indispensable amino acid of cell utilization and non-essential amino acid there is suitable ratio.
(2) trace element: comprise magnesium iron copper manganese nisiloy vanadium molybdenum selenium tin zinc etc., these trace elements have the various kinds of cell vital processes such as adjusting energy metabolism widely, albumen be synthetic, be the indispensable part of substratum, but its rational scope also need determining of experiment.As Mg: ATP enzyme, kinases etc. are played to activation.Iron is the prothetic group of enzyme and protoheme, is the integral part of respiratory chain in plastosome; Cu is the prothetic group of superoxide-dismutase, is the integral part of respiratory chain in plastosome.Selenium: the integral part of glutathione peroxidase, is present in many skins chain with the form of seleno-cysteine.Zn is the prothetic group of some enzymes.
(3) VITAMIN: mainly play the part of the role of coenzyme prothetic group, essential.Choline and thanomin are the important compositions that forms cytolemma, and putrescine can promote Chinese hamster ovary celI growth effectively.Folic acid is the important source material of tetrahydrobiopterin synthesis folic acid, and tetrahydrofolic acid (THFA) plays an important role in the biosynthesizing of nucleic acid and the biosynthetic process of protein.Vitamin H is the integral part of some special carboxylases, the building-up process of involved in sugar metabolism and lipid acid.
(4) other composition: lipid acid cell growth has promoter action.Yeast extract contains polypeptide and amino acid, and cell growth is favourable.
In addition,, outside substratum of the present invention, when configuration substratum, also to add following composition:
Regular Insulin: promote glucose and amino acid by cytolemma, be beneficial to absorption and the metabolism of cell; Promote the phosphorylation of the synthetic and Metabolic Intermediate of lipid and protein.Regular Insulin maintains cell in fission process neutralization and plays an important role at healthy physiological metabolism state aspect.
Glutamine: the contained nitrogen of glutamine is the source of purine and pyrimidine in nucleic acid, is cell nucleic acid and protein essential amino acid, and glutamine is also utilized by cell as the energy and carbon source.Substratum is not containing this thing, but necessary interpolation before using.
Glucose: the main source of cellular energy, add on demand.
The composition of basic medium provided by the invention:
First part: basic composition
| Composition | Content: g/L substratum |
| Sodium-chlor | 6－6.5 |
| Repone K | 0.5－0.5 |
| Sodium.alpha.-ketopropionate | 0.462－1 |
| Nitrocalcite | 0.288－0.4 |
| Sodium bicarbonate | 1.464－2 |
| D-Glucose | 5.16－15 |
| Sodium phosphate dibasic | 0.156－0.7 |
The second part: amino acid
| Composition | Content: g/L substratum |
| ALANINE | 0.03738－0.218673 |
| Glycine | 0.0495－0.289575 |
| ILE | 0.3978－2.32713 |
| L-Trp | 0.090648－0.5302908 |
| L-Leu | 0.58992－3.451032 |
| Altheine | 0.62244－3.641274 |
| L-Phe | 0.060432－0.3535272 |
| TYR | 0.2328－1.36188 |
| L-Aspartic acid | 0.558－3.2643 |
| Pidolidone | 0.06174－0.361179 |
| L-arginine | 0.7734－4.52439 |
| Cys | 0.14748－0.862758 |
| L-Histidine | 0.113232－0.6624072 |
| L-PROLINE | 0.14496－0.848016 |
| 1B | 2.4084－14.08914 |
| Serine | 0.6684－3.91014 |
| L-threonine | 0.9－5.265 |
| L-Methionine | 0.1842－1.07757 |
| Valine | 0.5268－3.08178 |
| Hydroxyl L-PROLINE | 0.5－2.925 |
The 3rd part: trace element:
| Composition | Content: g/L substratum |
| Magnesium sulfate | 0.2748－1.60758 |
| Ferric sulfate | 0.516－3.0186 |
| Copper sulfate | 0.00144－0.008424 |
| Manganous chloride tetrahydrate MnCl2 | 0.0000050.00002925 |
| Single nickel salt, NiSO4 | 0.00000033－1.9305E-06 |
| Water glass Na2SiO3 | 0.000284－0.0016614 |
| Sodium metavanadate NaVO3 | 0.00000146－0.000008541 |
| Sodium orthomolybdate NaMoO4 | 0.00000242－0.000014157 |
| Sodium Selenite Na2SeO3 | 0.0000173－0.000101205 |
| Tin protochloride SnCl2 | 0.00000028－0.000001638 |
| Zinc sulfate ZnSO4 | 0.39564－2.314494 |
The 4th part: VITAMIN:
The 5th part: other composition:
| Composition | Content: g/L substratum |
| 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) | 4－6 |
| Pluronic F-68F(Pluronic F-68) | 1－2 |
| Linolic acid | 0.0003528－0.00206388 |
| Yeast extract (DMV) | 3－10 |
The composition of supplemented medium provided by the invention:
First part: fundamental component
| Composition | Content: g/L substratum |
| Sodium.alpha.-ketopropionate | 5.67－10.773 |
| Nitrocalcite | 4.32－8.208 |
| D-Glucose | 100－190 |
| Sodium phosphate dibasic | 3.24－6.156 |
Second section: amino acid
| Composition | Content: g/L substratum |
| ALANINE | 0.5607－1.06533 |
| Glycine | 0.7425－1.41075 |
| ILE | 7.5－14.25 |
| L-Trp | 1.93－3.667 |
| L-Leu | 8.8488－16.81272 |
| Altheine | 9.3366－17.73954 |
| L-Phe | 1.3－2.47 |
| TYR | 4.3－8.17 |
| L-Aspartic acid | 8.3715.903 |
| Pidolidone | 0.9261－1.75959 |
| L-arginine | 9－17.1 |
| Cys | 3－5.7 |
| L-Histidine | 1.69848－3.227112 |
| L-PROLINE | 1.2－2.28 |
| 1B | 36.126－68.6394 |
| Serine | 8－15.2 |
| L-threonine | 13.5－25.65 |
| L-Methionine | 4－7.6 |
| Valine | 17－32.3 |
| Hydroxyl L-PROLINE | 15－28.5 |
Part III: trace element
| Composition | Content: g/L substratum |
| Magnesium sulfate | 0.423－0.8037 |
| Ferric sulfate | 3.5－6.65 |
| Copper sulfate | 0.0216－0.04104 |
| Manganous chloride tetrahydrate MnCl2 | 0.000075－0.0001425 |
| Single nickel salt NiSO4 | 0.00000495－0.000009405 |
| Water glass Na2SiO3 | 0.00426－0.008094 |
| Sodium metavanadate NaVO3 | 0.0000219－0.00004161 |
| Sodium orthomolybdate NaMoO4 | 0.0000363－0.00006897 |
| Sodium Selenite Na2SeO3 | 0.0002595－0.00049305 |
| Tin protochloride SnCl2 | 0.0000042－0.00000798 |
| Zinc sulfate ZnSO4 | 5.9346－11.27574 |
Part IV: VITAMIN
Part V: other composition
| Composition | Content: g/L substratum |
| Linolic acid | 0.005292－0.0100548 |
| Yeast extract | 30－100 |
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets discloses can with any composition forms use, each feature disclosing in specification sheets, can anyly provide the alternative characteristics of identical, impartial or similar object to replace.Therefore apart from special instruction, the feature disclosing is only the general example of equalization or similar features.
Major advantage of the present invention is:
1, the serum-free basic medium that is exclusively used in Chinese hamster ovary celI provided by the invention, the Chinese hamster ovary celI density of its cultivation exceedes in the world general JRH substratum.
2, in large scale culturing, contriver, by giving the feed supplement special with Chinese hamster ovary celI in the suitable time, increases considerably the density of Chinese hamster ovary celI and expression amount, and fermented liquid Chinese traditional medicine protein content has reached the level of 1.7 grams/L.
3, serum free medium provided by the invention and feed supplement thereof are not added as source using any commercial product culture medium, have high application and economic worth, significant to the level of the extensive animal cell culture technology of raising China.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all per-cent and umber by weight.
Unit in percent weight in volume in the present invention is well-known to those skilled in the art, for example, refer to the weight of solute in the solution of 100 milliliters.
Unless otherwise defined, the same meaning that all specialties that use in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
The JRH substratum using in the embodiment of the present invention, purchased from Sigma (St Louis, MO, USA), is Excel325 serum free medium (catalog number (Cat.No.) 24340C); Invitrogen substratum, purchased from Invitrogen (Grand Island, NY, USA), is CD-CHO serum free medium (catalog number (Cat.No.) 10743-029).JRH supplemented medium, purchased from Sigma (St Louis, MO, USA), is CHO reactor supplemented medium (catalog number (Cat.No.) C1615); Invitrogen supplemented medium, purchased from Invitrogen (Grand Island, NY, USA), is CD-CHO50X supplemented medium (the acid fluid infusion of catalog number (Cat.No.) 001-0042CD-CHO50X, the fluid infusion of 001-0084CD-CHO50X alkalescence).
Embodiment 1
Preparation is for the basic medium of Chinese hamster ovary celI
One, preparation basic medium:
1. composition
Match gold base CHO substratum (preparing by table one)
NaHCO
3?1.6g/L
L-glutaminate 0.4344g/L
Glucose 6.4g/L
Regular Insulin (10mg/ml) 50ul is: 0.5mg/L
2. filter is prepared
Ultrapure water: more than water making machine terminal resistance value > 17 megohms, confirmation balance is normally worked, and balance bubble is adjusted.
Select filter, applicable filtering material: the hydrophilic filter core of cartridge type or dull and stereotyped filter membrane according to culture volume.
Cylindrical filter cartridge: 0.22um, size is applicable to sleeve, makes integrity detection
Dull and stereotyped filter membrane: 0.45um, the each a slice of 0.22um, fully moistening
Filter sterilizing: 122 DEG C, 30 minutes
3. preparation
Additional match gold base CHO substratum, glucose and Glutamine dissolve with the ultrapure water of final volume 90%, fully stir half an hour, add NaHCO3, keep stirring 1 hour after adding again, and add Regular Insulin, are settled to required final volume and keep stirring 10-15 minutes.
Sterile Filtration, is undertaken by core strainer or flat-panel filter Standard operation procedure SOP.
Table one is matched the composition of gold base substratum
Embodiment 2
Preparation is for the supplemented medium of Chinese hamster ovary celI
One, preparation fed-batch medium:
1. composition
Match golden supplemented medium (in the ratio preparation of table two)
Glucose 24.4g/L
2. filter is prepared
Ultrapure water: more than water making machine terminal resistance value > 17 megohms, confirmation balance is normally worked, and balance bubble is adjusted.
Select filter, applicable filtering material: the hydrophilic filter core of cartridge type or dull and stereotyped filter membrane according to culture volume.
Cylindrical filter cartridge: 0.22um, size is applicable to sleeve, makes integrity detection
Dull and stereotyped filter membrane: 0.45um, the each a slice of 0.22um, fully moistening
Filter sterilizing: 122 DEG C, 30 minutes
3. preparation
Additional match gold supplemented medium and glucose dissolve with the ultrapure water of final volume 90%, fully stir half an hour, are settled to required final volume and keep stirring 10-15 minutes.
Sterile Filtration, is undertaken by core strainer or flat-panel filter Standard operation procedure SOP.
Table two is matched golden supplemented medium preparation
Embodiment 3
Base culture base Chinese hamster ovary celI
Test method: batch cultivation (not carrying out feed supplement)
Testing sequence: cultivate in checking at this, do not carry out feed supplement, after inoculation, cell survival rate is reduced to 50% left and right for cultivating terminal.
Culture environment: 37 DEG C, 5% carbonic acid gas
Shake-flask culture process: in super clean bench respectively to the basic medium and the JRH substratum that add the embodiment 1 having filtered in two 125ml shaking flasks.Access seed (counting the 0th day), the whole density that makes cell is 0.5x10
6, cumulative volume 30ml.Cell enters after date in plateau and starts cooling, makes cell transfer expressing protein to by growth conditions.
Cultivation results: at the 7th day of the golden culture medium culturing of use match, it is the highest that cell density reaches: 5.3x10
6, survival rate 99.1%.And with the cell density of JRH base culture base be 3.9x10
6, survival rate 97.5%, JRH basic medium reached high-density 4.1x10 at the tenth day
6, survival rate 93.5%.
| Item compared | The basic medium of embodiment 1 | JRH basic medium |
| High-cell density (cell/ml) | 5.3x10 6 | 3.9x10 6 |
| Activity when high-cell density | 99.1% | 97.5% |
| Albumen titre (g/L) | 0.4 | 0.2 |
Embodiment 4
Chinese hamster ovary celI nutrient solution is carried out to feeding culture (adding supplemented medium):
Test method: feeding culture (adding supplemented medium)
Testing sequence: in this culture experiment, the feed supplement of carrying out at Growth of Cells to logarithmic phase mid-term, in the mid-term of plateau, the operation of lowering the temperature starts expressing protein to cell, and survival rate is reduced to 50% left and right for cultivating terminal.
Culture environment: 37 DEG C, 5% carbonic acid gas
Shake-flask culture process: respectively to the basic medium, JRH and the Invitrogen substratum that add the embodiment 1 having filtered in three 125ml shaking flasks, making cell cumulative volume after inoculation is 30ml in super clean bench, and when inoculation, cell density is all: 0.5x10
6cell/ml.Carry out feed supplement at Growth of Cells to logarithmic phase mid-term (the 7th day), add the supplemented medium 750 μ l left and right of concentrated embodiment 2, not too large variation of cell culture fluid volume (sampling and some volumes of evaporation meeting loss).Cell enters after date in plateau and starts cooling, makes cell transfer expressing protein to by growth conditions.
Result
1. basic medium:
The result of base culture base Chinese hamster ovary celI of the present invention shows, Growth of Cells is vigorous, rapid, cytoactive is high, in cultivation, can reach very high cell density, in contriver's experiment, the highest cell density has exceeded domestic conventional JRH substratum 36%, albumen titre exceedes one times of JRH substratum, so the recipe ratio JRH of basic medium provided by the invention is more reasonable, more can promote the growth of cell in cultivation.
2. supplemented medium
Supplemented medium of the present invention adds after the logarithmic phase CHO nutrient solution in mid-term, and cell can reach higher cell density, in the time of high cell density, can also keep very high activity, for next step cooling expressing protein provides good guarantee.
Compare by experiment, match golden supplemented medium and can reach higher cell density than JRH, Invitrogen, improve 74% than JRH, higher by 76% than Invitrogen, the protein content of expression is higher by 212.5% than JRH, higher by 283.3% than Invitrogen.
The foregoing is only preferred embodiment of the present invention, not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is to be broadly defined in the claim scope of application, any technology entity or method that other people complete, if defined identical with the claim scope of application, also or a kind of change of equivalence, be all covered by among this claim scope being regarded as.
Claims (6)
1. for a basic medium for Chinese hamster ovary cell, it is characterized in that, described basic medium contains following fundamental component:
6-6.5g/L sodium-chlor;
0.5-0.5g/L Repone K;
0.462-1g/L Sodium.alpha.-ketopropionate;
0.288-0.4g/L nitrocalcite;
1.464-2g/L sodium bicarbonate;
5.16-15g/L D-Glucose; With
0.156-0.7g/L Sodium phosphate dibasic, in the cumulative volume of described basic medium;
Described basic medium also contains 10-54g/L amino acid, 1.2-7.0g/L trace element, 0.3-1.6g/L VITAMIN and 3-10g/L yeast extract;
Described amino acid (g/L) is:
Described trace element (g/L) is:
Described VITAMIN (g/L) is:
2. for a supplemented medium for Chinese hamster ovary cell, it is characterized in that, described supplemented medium contains following fundamental component:
5.67-10.773g/L Sodium.alpha.-ketopropionate;
4.32-8.208g/L nitrocalcite;
100-190g/L D-Glucose; With
3.24-6.156g/L Sodium phosphate dibasic, in the cumulative volume of described supplemented medium;
Described supplemented medium also contains 153-300g/L amino acid, 10-19g/L trace element, 5.5-11g/L VITAMIN and 30-100g/L yeast extract;
Described amino acid (g/L) is:
Described trace element (g/L) is:
Described VITAMIN (g/L) is:
3. the purposes of basic medium claimed in claim 1, is characterized in that, for cultivating Chinese hamster ovary cell.
4. the purposes of supplemented medium claimed in claim 2, is characterized in that, for cultivating Chinese hamster ovary cell.
5. a cultural method for Chinese hamster ovary cell, is characterized in that, described method comprises step:
(a) in basic medium as claimed in claim 1, cultivate Chinese hamster ovary cell; With
(b) add supplemented medium as claimed in claim 2 to cultivate Chinese hamster ovary cell at Growth of Cells to logarithmic phase.
6. cultural method as claimed in claim 5, is characterized in that, in step (a), with 0.5x10
6in the initial cultivation of density; In step (b), cultivating 3-5 days cell density 4-7x10
6time give feed supplement.
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| BR112021025769A2 (en) | 2019-12-06 | 2022-04-12 | Regeneron Pharma | Anti-vegf protein compositions and methods for their production |
| CN115125278B (en) * | 2022-08-25 | 2022-11-18 | 山东合成远景生物科技有限公司 | Feed supplement liquid for producing polysialic acid and preparation method of polysialic acid |
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| US20040171152A1 (en) * | 1996-10-10 | 2004-09-02 | Invitrogen Corporation | Animal cell culture media comprising non-animal or plant-derived nutrients |
| CN101195816A (en) * | 2007-12-28 | 2008-06-11 | 天津百若克医药生物技术有限责任公司 | Gonad cell amplification culture medium of Chinese hamster and uses thereof |
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|---|---|---|---|---|
| US20040171152A1 (en) * | 1996-10-10 | 2004-09-02 | Invitrogen Corporation | Animal cell culture media comprising non-animal or plant-derived nutrients |
| CN101195816A (en) * | 2007-12-28 | 2008-06-11 | 天津百若克医药生物技术有限责任公司 | Gonad cell amplification culture medium of Chinese hamster and uses thereof |
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| "无血清细胞培养基的主要补充因子及其研究进展";陈锋;《海峡药学》;20061231;第18卷(第4期);第12页 右栏 第1段 * |
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